Fig. 3.

LtaS_S218P variant with mutated cleavage site shows reduced processing but retains activity. (A) S. aureus strains used for complementation analysis contained the chromosomal copy of the ltaS gene under IPTG-inducible spac promoter control, and the LtaS variant under investigation (X) was expressed from an Atet-inducible promoter at an ectopic chromosomal location. (B) Bacterial growth curves. Expression of WT LtaS and the LtaS_S218P variant was induced after removal of IPTG by the addition of Atet. As a negative control, an S. aureus strain containing the empty pitet vector (no insert) was used, and growth of all cultures was monitored by determining OD₆₀₀ readings at the indicated time points. Cultures were back diluted 1:100 in fresh medium at the 4-h time point to maintain cultures in the logarithmic growth phase. (C) Detection of LTA by Western blotting. At the 4-h time point, 1-ml culture aliquots were removed and samples prepared and analyzed by Western blotting. A mouse monoclonal anti-LTA antibody (clone 55; Hycult Biotechnology) was used for the detection of LTA in the cell-associated fraction. (D) Detection of LtaS by Western blotting. At the 4-h time point, 1-ml culture samples were removed and supernatant (sup.) and whole-cell (cell) samples prepared as described in the legend to Fig. 1C. A star indicates the full-length LtaS protein, a square the cleaved eLtaS protein, and a filled circle the unspecific Sbi protein band. (E) Schematic representation of WT LtaS and the LtaS_S218P variant. Numbers refer to amino acid positions, and arrows indicate main cleavage sites identified by mass spectrometry analysis. The proline substitution in the LtaS_S218P mutant is shown as a bold gray letter. S. aureus strains ANG513, ANG514, and ANG1246 were used for this experiment.